bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Elucidating the diversity of microeukaryotes and epi-endophytes in the brown 2 algal holobiome 3 4 5 Marit F. Markussen Bjorbækmo1,2*, Juliet Brodie2, Ramiro Logares3, Stephanie 6 Attwood2, Stein Fredriksen4, Janina Fuss1, Kamran Shalchian-Tabrizi1, Anders Wold- 7 Dobbe1, Anders K. Krabberød1, David Bass2,5* 8 9 10 1. University of Oslo, Department of Biosciences, Section for Genetics and 11 Evolutionary Biology (EVOGENE), N-0316, Oslo, Norway 12 2. The Natural History Museum (NHM), Department of Life Sciences, London SW7 13 5BD, United Kingdom 14 3. Institut de Ciències del Mar (CSIC), Department of Marine Biology and 15 Oceanography, ES-08003, Barcelona, Catalonia, Spain 16 4. University of Oslo, Department of Biosciences, Section for Aquatic biology and 17 Toxicology (AQUA), N-0316, Oslo, Norway 18 19 5. Centre for Environment Fisheries and Aquaculture Science (CEFAS), Weymouth, 20 Dorset DT4 8UB, United Kingdom 21 22 23 * Correponding authors: 24 David Bass 25 e-mail: [email protected] 26 Phone: 27 28 Marit F. Markussen Bjorbækmo 29 e-mail: [email protected] 30 Phone: 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 31 Abstract 32 Background 33 Brown algae (Phaeophyceae) are essential species in coastal ecosystems where they 34 form kelp forests and seaweed beds that support a wide diversity of marine life. Host- 35 associated microbial communities are an integral part of phaeophyte biology. The 36 bacterial microbial partners of phaeophytes have received far more attention than 37 microbial eukaryotes. The pre-requisite to understand the ecology of phaeophytes and 38 their host-associated microbes is to know their diversity, distribution and community 39 dynamics. To our knowledge, this is the first study to investigate phaeophyte-associated 40 eukaryotes (the eukaryome) using broadly targeting ‘pan-eukaryotic’ primers and high 41 throughput sequencing (HTS). Using this approach, we aimed to unveil the eukaryome 42 of seven large common phaeophyte species. We also aimed to assess whether these 43 macroalgae harbour novel eukaryotic diversity and to ascribe putative functional roles 44 to the host-associated eukaryome, based on taxonomic affiliation and phylogenetic 45 placement. 46 47 Results 48 Our sequence dataset was dominated by phaeophyte reads, from the host species and 49 potential symbionts. We also detected a broad taxonomic diversity of eukaryotes in the 50 phaeophyte holobiomes, with OTUs taxonomically assigned to ten of the eukaryotic 51 major Kingdoms or supergroups. A total of 265 microeukaryotic and epi-endophytic 52 operational taxonomic units (OTUs) were defined, using 97% similarity cut off during 53 clustering, and were dominated by OTUs assigned to Stramenopiles, Alveolata and 54 Fungi. C Almost one third of the OTUs we detected have not been found in previous 55 molecular environmental surveys, and represented potential novel eukaryotic diversity. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 56 This potential novel diversity was particularly diverse in phylogenetic groups 57 comprising heterotrophic and parasitic organisms, such as labyrinthulids and 58 Oomycetes, Cercozoa, and Amoebozoa. 59 60 Conclusions 61 Our findings provide important baseline data for future studies of seaweed-associated 62 microorganisms, and demonstrate that microeukaryotes and epi-endophytic eukaryotes 63 should be considered as an integral part of phaeophyte holobionts. The potential novel 64 eukaryotic diversity we found and the fact that the vast majority of macroalgae in 65 marine habitats remain unexplored, demonstrates that brown algae and other seaweeds 66 are potentially rich sources for a large and hidden diversity of novel microeukaryotes 67 and epi-endophytes. 68 69 70 71 Keywords [3-10] 72 Symbiome, epiphyte, endophyte, protist, microeukaryote, eukaryome, Phaeophyceae, 73 seaweed, holobiont, fungi 74 75 76 77 Background 78 Seaweeds are essential to the health of our planet, providing core ecosystem services, 79 and food and shelter to a wide diversity of marine life, from microbes to mammals, [1, 80 2 and references therein]. Brown algal seaweeds (Phaeophyceae) are Stramenopiles, a 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 81 radiation of eukaryotes which is phylogenetically distant from red- and green algal 82 seaweeds (Archaeplastida; Chlorophyta and Rhodophyta). The phaeophytes are one of 83 the most diversified groups of benthic algae and comprise approximately 2000 species, 84 encompassing complex multicellular species which have diversified and evolved since 85 the Mesozoic Era ~250 MYA [2, 3]. Large phaeophytes, especially those in the orders 86 Laminariales, Fucales and Tilopteridales are dominant members of intertidal and 87 shallow subtidal ecosystems worldwide, where they function as ecosystem engineers 88 forming complex underwater forests. These coastal ecosystems are some of the most 89 productive habitats on the planet [4], where seaweed beds and kelp forests are 90 indispensable biodiversity hotspots, which also provide habitat and nursery for a wide 91 range of taxa, including many commercially important species [5]. Phaeophytes have 92 been shown to accommodate up to 100.000 individuals of different invertebrate species 93 per square metre [6]. 94 Phaeophytes and other seaweeds also harbour a wide diversity of microbial 95 epibionts and endobionts, comprising eukaryotes [7-9], prokaryotes [10-12] and viruses 96 [13-16]. These, together with the host, are collectively referred to as the seaweed 97 holobiont and can be considered as a localized ecosystem living on and in a host [10, 98 17-19]. It is increasingly recognized that host-associated microbial communities 99 (microbiomes) are an integral part of the host biology, exerting diverse and strong 100 influences on their hosts [19, 20]. While the term microbiome usually refers mostly or 101 exclusively to bacteria, it is important to consider the whole microbial symbiome, 102 including microbial eukaryotes (microeukaryotes) and larger epiphytic, epizootic, and 103 endophytic symbionts, to enable a comprehensive understanding of holobiont 104 functioning [19, 21]. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 105 Symbiotic interactions in its broadest sense refers to all intimate ecological 106 interactions between two species [22]. According to the fitness effect on the members 107 involved in the symbiotic relationship, the associations cover a wide spectrum of 108 interactions along the gradual continuum between positive mutualism and negative 109 parasitism. Some symbiotic interactions are obligate while other are more temporary 110 and fluctuating, and the nature of the interaction can vary depending on the symbiont’s 111 interactions with its hosts, other symbionts and environmental conditions [20]. To date, 112 bacterial symbionts of phaeophytes have received most study [e.g., 10, 11, 12, 23], and 113 have been shown to represent complex and highly dynamic relationships that can have 114 everything from fundamental to detrimental effects on their hosts [10, 24-28]. 115 Microeukaryotes in the phaeophyte symbiome are far less well known. Only a 116 small number of studies have investigated these associations, mostly using traditional 117 culturing/cell isolation methods or targeted molecular approaches. These studies 118 demonstrate that a broad taxonomic diversity of microeukaryotes are associated with 119 phaeophytes, including surface dwelling heterotrophic diatoms, dinoflagellates and 120 ciliates [8], ‘naked amoeba’ [29], epiphytic and endophytic diatoms [30-32], and green 121 algal endophytes [33], in addition to parasitic or saprotrophic labyrinthulids [34, 35], 122 oomycetes [7, 36, 37], phytomyxids [38-40] and fungi [9, 41-44]. The nature of these 123 microeukaryote-host relationships is mostly unknown, although some symbionts can 124 have detrimental effects on their macroalgal hosts, for example phytomyxids [38-40], 125 oomycetes [7, 36, 37] and chytridiomycete fungi [41]. Other microeukaryotes are 126 suspected